The purpose of these studies is to establish a better understanding of the energy metabolism in tissues, in vivo. Towards this goal, the laboratory concentrates on the use of non-invasive and non-destructive techniques to evaluate the biochemical and physiological function of the heart and skeletal muscle with regard to energy metabolism. The following major findings were made over the last year: 1) A mathematical network model is being developed to create a consensus model of mitochondrial function and oxidative phosphorylation. Using this approach we have identified the first step of NADH oxidation in oxidative phosphorylation as problematical in using in vitro data to model this process. Based on in vitro data, the product inhibition of the mMolar concentration of NADH in the matrix should completely inhibit most of the dehydrogenases of Kreb?s cycle. Clearly this does not occur, in vivo and approaches are being developed to better understand the metabolism of NADH in the mitochondrial matrix. 2) The binding of NADH in the mitochondria matrix was evaluated using fluorescence lifetime and emission spectroscopy. Binding of NADH to the membranes or matrix can be followed by the blue spectral shift and enhanced lifetime of the bound form of NADH. Using two preparations, intact mitochondria and isolated mitochondria membranes we have found that the binding affinity of NADH increases by nearly 3 orders of magnitude in isolated membranes. This implies that the micro-environment in the mitochondria matrix is dramatically different than in a simple membrane or isolated protein preparation. The amount of bound NADH, followed by these optical methods, was directly proportional to the rate of NADH oxidation in both preparations, implying that the binding site(s) are critically involved in the oxidation of NADH in both preparations. These data suggest that some of the discrepancies between in vitro biochemical determinations and in vivo measurements concerning NADH oxidation might be related to the specific structures or associations in the matrix, lost in protein isolation. In addition, the low binding affinity of NADH in the matrix results in a near linear relationship between total matrix NADH and matrix NADH fluorescence. This later result suggests that mitochondrial NADH fluorescence measurements, in vivo, are reflecting the overall concentration of mitochondrial NADH. 3) Minimally invasive, two photon excitation fluorescence microscopy (TPEFM) in intact animals is being used to study sub-cellular metabolic processes within cells under normal in vivo conditions. This optical microscopy approach provides an important link between classical cell biology and whole animal physiology studies permitting the evaluation of cellular events in the intact animal. In the mouse skeletal muscle, in vivo, we have shown that TPEFM can non-invasively fiber type the individual cells, observe the distribution and blood flow through capillaries and directly observe the distribution and redox state of perivascular, perinuclear, and fibrillar mitochondria under a variety of conditions, in vivo. In our initial ischemia-reperfusion studies to determine the sub-celllular events associated with this alteration in metabolism, we observed the important influences of tissue absorbance changes that need to be corrected for in interpreting these data in vivo. Currently studies are underway to investigate the role of perivascular mitochondria in the control of regional blood flow in mouse skeletal muscle. 4) A screening of the mitochondria proteome and phosphoproteome is being undertaken using 2D gel electrophoresis and mass spectroscopy techniques. The initial studies have concentrated on identifying the phosphoproteome of the mitochondria with phosphorylation sites found in each of the complexes of oxidative phosphorylation and extensive phosphorylation sites in numerous metabolic enzymes involved in substratte oxidation as well as other regulatory enzymes such as superoxide dismutase. Currently, the effect of extramitochondrial calcium and metabolic state of the animal prior to isolation of mitochondria are being investigated.